Plan apochromat 20x 0.75 objective

Ex vivo experiments were conducted on cells grown on Lab-Tek chambered coverglass (Nalgen Nunc International) and maintained under standard culture conditions (37°C, 5% CO₂). Spheroids were recovered by pipetting just before imaging and deposited in the Lab-Tek well containing cell culture medium. Images were acquired on a Zeiss dynascope confocal microscope using a Plan-Apochromat 20X / 0.75 objective. Images were analysed with the Zen software provided by Zeiss and a median filter (3/3/1) was applied to all images. At least 10 mitotic cells were simultaneously followed and three independent experiments were conducted.

All surgical procedures are approved by NYU- School of Medicine IACUC. Images were acquired with Zeiss Plan-Apochromat 20X/0.75 objective on ZEISS LSM 510. Intravital imaging was conducted as previously described (
Velázquez
et al., 2008). In brief, upon completion of the surgery, animals were placed with the abdominal side down on a custom-made stage insert (Ludl Electronic Produces, Hawthorne, NY, USA), in which a coverslip (No. 1.5: 0.16–0.19 mm thick) was mounted near the center and narrow strips of paper (1.5 mm × 1.5 cm) was glued. These strips of paper provided friction that help immobilize the tissue for imaging.
The stage insert was then placed on an inverted microscope stage and imaged. For time-lapse image acquisition, 1 volume was collected every 30 s, with z-slices acquired every 5 μm. The maximum depth acquired over a 30-s interval with a single-photon light source was 20 μm utilizing a 2 μs dwell time. The animal is maintained at 37°C in an environmental chamber with supplemental medical-grade oxygen supplied via a nose cone. To visualize blood perfusion, mice were treated with an intravenous dose of low molecular weight 10 kDa dextran conjugated to Alexa-647 fluor (Molecular Probes) while imaging. Animals were euthanized at the end of each experiment.